CN101153057A - Protein for improving plants fastness and accelerating vegetation and encoding gene thereof - Google Patents
Protein for improving plants fastness and accelerating vegetation and encoding gene thereof Download PDFInfo
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- CN101153057A CN101153057A CNA2006101527008A CN200610152700A CN101153057A CN 101153057 A CN101153057 A CN 101153057A CN A2006101527008 A CNA2006101527008 A CN A2006101527008A CN 200610152700 A CN200610152700 A CN 200610152700A CN 101153057 A CN101153057 A CN 101153057A
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Abstract
The present invention relates to protein for improving disease resistance and enhancing plant growth, and the encoding gene. The present invention provides a type of protein PEAt1 separated from chain Alternaria. The protein has the function of improving disease resistance and enhancing plant growth. The present invention also relates to the preparation method and uses of the protein and multi nucleotide acid, and the expression carrier and transformer constructed by the gene of the multi nucleotide acid.
Description
Technical field:
The invention belongs to biological technical field, relate to a kind of albumen that promotes plant-growth and improve disease resistance of plant from alternaric bacteria (Alternaria spp) bacterium.The invention still further relates to this proteic nucleotide sequence of coding, and comprise expression vector and the transformant that this nucleotide sequence makes up.
Background technology:
Alternaric bacteria (Alternaria spp) has parasitic and saprophytic property, is the important plant pathogenic fungi of a class, causes plurality of plant diseases, produces the Plant diseases that causes for alternaric bacteria, and existing people did many researchs.The proteinoid that promotes plant-growth and improve the disease resistance of plant effect that has that extracts from the various plants pathogenic fungi is disclosed among the Chinese patent ZL 01 128666.0 (Qiu Dewen " plant with multi-functional true mycoprotein goods and preparation method thereof ").The albumen and the relevant information thereof of separation and purification from rice blast fungus are disclosed in the China number of applying for a patent CN200510011580.5 (Qiu Dewen etc. " promote the protein and the synthetic gene thereof of plant-growth and raising disease resistance of plant ").But so far not clearly from alternaric bacteria (Alternaria spp) albumen of separation and purification have function and the relevant information thereof that promotes plant-growth and improve disease resistance of plant.In GenBank, do not find to have and promote plant-growth and improve the alternaric bacteria albumen of plant disease-resistant sexual function and the information of nucleotide sequence.
Summary of the invention:
The objective of the invention is to seek and have the functional protein that promotes plant-growth and improve disease resistance of plant; Another object of the present invention is according to this proteic aminoacid sequence, and this proteic gene of method acquisition coding by RT-PCR with its construction of expression vector and transformant, is used to promote the albumen of plant-growth and raising disease resistance of plant with preparation.
The present invention's separation and purification from Alternaria tenuissima bacterium (Alternaria tenuissima) has gone out a kind of protein, and this proteic gene of encoding is 624bp, is made up of 207 amino acid, and molecular weight is 22590Da.By Genbank gene pool comparison, know this kind albumen and be undiscovered a kind of albumen in alternaric bacteria (Alternaria).This Argine Monohydrochloride is formed and formed similarity with the proteic amino acid of the nascent polypeptide prozyme (nascent polypeptide-associated complex (NAC) GenBank accession number CH445335) of Phaeosphaeria nodorum SN15 is 91.98%.
Because this kind dietary protein origin, has the effect that promotes plant-growth and improve disease resistance of plant in alternaric bacteria, with this albumen called after PEAt1.For confirming the proteic function of above-mentioned isolating PEAt1, the inventor has carried out experimental study to it to the promoter action of plant root growth with to the restraining effect of tobacco mosaic virus disease.Confirm that the isolating PEAt1 albumen of the present invention has the function that promotes plant-growth and improve disease resistance of plant.This albumen can be used for preparing the goods that promote plant-growth and improve disease resistance of plant.
Be connected the recombinant plasmid that obtains with vector plasmid with this proteic gene of encoding and import the host microorganism cell, can obtain containing the transformant of this recombinant plasmid; By cultivating this transformant, the expressing protein that inducing culture obtains has the plant-growth of promotion and improves the disease resistance of plant effect, can be used for preparing the goods that promote plant-growth and improve disease resistance of plant.Described transformant, the host microorganism cell is all optional from intestinal bacteria and Pichia yeast.
Embodiment:
The proteic isolation and purification of embodiment 1 PEAt1
Cultivate alternaric bacteria with the PDA liquid nutrient medium, under 28 ℃, 180r.min
-1, constant-temperature shaking culture 2d, the mycelium that suction filtration obtains is got the mycelia piece and become fine powder with liquid nitrogen grinding in mortar, adds the phosphoric acid extraction damping fluid rapidly, after boiling water bath boils 20min, puts in the mixture of ice and water and lowers the temperature, 12000r.min under 4 ℃ of conditions
-1Centrifugal 15min, supernatant liquor filters with millipore filter (Ф=0.22 μ m), obtains extracting solution.Directly adding ammonium sulfate to final concentration in protein extract is 85%, continue to get precipitation with the centrifugal 30min of 5000rpm behind the magnetic stirrer 30min, to precipitate again with extracting the damping fluid dissolving, be to add-20 ℃ of precooling dehydrated alcohols ,-20 ℃ of precipitation 30min at 4: 1 according to the ratio of sample and ethanol volume, 4 ℃ of centrifugal 10min of 5000rpm, get precipitation, ice bath is dried to alcohol-free smell, after the dissolving of anionresin initial buffer liquid, 4 ℃ of centrifugal 30min of 13000r/min obtain protein crude extract.Adopt anionresin low salt buffer dialysed overnight, obtain crude protein solution.Obtain purifying protein by ion exchange chromatography and molecular sieve gel chromatography then.
Embodiment 2 PEAt1 albumen are to the promoter action of plant root growth
Wheat seed soaked seed respectively be diluted to 3 μ g.ml at purifying protein
-1, 2 μ g.ml
-1, 1 μ g.ml
-1The aqueous solution in, with distilled water seed soaking as negative control.Soaking seed after 8 hours is tiled in seed in the culture dish in 28 ℃ of illumination box illumination cultivation, and adds an amount of distilled water to keep humidity.Cultivate random measurement after 7 days and respectively handle the main root length of wheat, each handle measure 30 parallel and average.
As seen from Table 1, growth still has promoter action to wheat root when protein concentration is 1 μ g/ml.Root length is being 3 μ g.ml in activator concentration
-1, 2 μ g.ml
-1, 1 μ g.ml
-1The time, the long comparison of wheat root you can well imagine high 12.29%, 11.7%, 8.06% according to component.
The variation that table 1 different concns protein solution is long to wheat root
| Contrast | 3μg.ml -1 | 2μg.ml -1 | 1μg.ml -1 |
| 10.17±0.85A | 11.42±0.325B | 11.36±0.552B | 10.99±0.4327AB |
Embodiment 3 PEAt1 albumen are to the restraining effect of tobacco mosaic virus (TMV)
Coral west cigarette is cultivated (20～27 ℃) in the insect protected greenhouse, plant grows to 8 leaves during the phase, adopts half leaf method, with PEAt1 albumen 2 μ g.ml
-1Soak blade 10min, compare with clear water, 6 leaves of every processing, every processing repeats 3 times.It is the culture dish of 15cm that blade after the processing places diameter, virus inoculation after preserving moisture under 25 ℃ 2 days.
Adopt conventional juice frictional inoculation method inoculation, get malicious source tobacco disease leaf and add 0.01mol/L phosphoric acid buffer (pH7.2) grinding (viral juice gravity is that the sick leaf of every gram adds the 50mL0.01moL/L phosphoric acid buffer).Blade after the processing adopts half leaf method equivalent inoculation TMV virus, writes down withered spot number after 3 days and is calculated as follows withered spot inhibiting rate.
PEAt1 albumen has higher restraining effect to the withered spot number of TMV on excised leaf, and the single leaf withered spot number between PEAt1 albumen and clear water are handled has significant difference, and withered spot inhibiting rate reaches 45～58%.
With two leaves about PEAt1 albumen and moisture other places Li Shanxi tobacco leaf sheet, measure withered spot size behind the equivalent virus inoculation after 3 days, then blade being placed changes behind 37 ℃ of following 24h in 25 ℃ of following incubations 3 days, measure withered spot size once more, write down the difference of twice withered spot size, calculate withered spot area increment rate.Show from measuring result, PEAt1 albumen can not only suppress withered spot size, expansion on blade has certain influence to TMV simultaneously, clear water is handled withered spot diameter increases 2.33mm, and the withered spot diameter that PEAt1 albumen is handled increases 1.51mm, withered spot expansion inhibiting rate is 54.3%, illustrates that PEAt1 albumen can suppress the further expansion of withered spot, effectively controls further developing of the tobacco virus state of an illness.
The proteic amino acid sequence analysis of embodiment 4 PEAt1
The PEAt1 albumen of purifying detects through SDS-PAGE, cuts single band behind trypsin digestion, measures proteinic peptide finger printing with ground substance assistant laser ionization-flight time mass spectrum (MALDI-TOF MS).Do liquid matter logotype (nanolc-ESI-IT-ms/ms) acquisition PEAt1 protein part peptide section aminoacid sequence (table 2) behind the enzymolysis respectively with trypsinase, curdled milk proteolytic enzyme, V8 enzyme.
Table 2 PEAt1 protein part peptide section aminoacid sequence
| The peptide section | Aminoacid sequence |
| PEAt1-1 PEAt1-2 PEAt1-3 PEAt1-4 | NILFVIN KPDVYKSPSSNTWIIFGEAK DIELVMQQASVSR ALKENDNDIVNSIMALSI RPKN I/L I/L FV I/L NQPDVYK |
The proteic gene clone of embodiment 5 coding PEAt1
Utilize Trizol reagent (Invitrogen) from the alternaric bacteria of fresh culture, to extract total RNA, obtain to contain cDNA first chain of oligo (dT) tail with the M-MuLV reversed transcriptive enzyme, aminoacid sequence design the forward primer 5 '-CCYAAGAACATYCTCTTYGTC-3 ' and the reverse primer 5 '-GTTGACg/tATATCRTTATCGTT-3 ' that at first utilize the mass spectrum order-checking to obtain utilize the method for RT-PCR to obtain the placed in the middle sequence of a segment length for 373bp; Utilize the sequence redesign primer that obtains to carry out 3 ' RACE, utilize reverse primer 5 '-GGCCACGCGTCGACTAGTAC (T)
16V-3 ' synthesizes cDNA first chain, utilize GSP1 5 '-AGGGCAAGGGCAAGGCTGTCG-3 ' and A1 to be one-sided PCR then, product with one-sided PCR is a template at last, uses GSP2:5 '-GGACGAGGAGGAGGAGGATGACG-3 ' and A2 5 '-GGCCACGCGTCGACTAGTAC-3 ' to carry out nest-type PRC; Utilize the sequence redesign primer placed in the middle that obtains to carry out 5 ' RACE.Utilize RT-primer5 '-CCTCGGCCTGAGCAAGCTGCTG-3 ' to synthesize cDNA first chain, 5 ' complete sequence adopts the method for Invitrogen GeneRacerkit to obtain; The nucleotide fragments that above-mentioned circulation is obtained splices nucleotide sequence that size is the entire reading frame of 624bp of back acquisition, with its called after peat1 gene (SEQ ID NO 1), comprise 4 peptide sections that obtain among the embodiment 4 in the aminoacid sequence of its supposition (SEQ IDNO 1), confirm that the peat1 gene that obtains is correct.
By Genbank gene pool comparison, know this kind albumen and be undiscovered a kind of albumen in alternaric bacteria (Alternaria).This Argine Monohydrochloride is formed and the proteic similarity of nascent polypeptide prozyme (nascentpolypeptide-associated complex (NAC) GenBank accession number CH445335) of Phaeosphaeria nodorum SN15 is 91.98%.
The structure and the expression of the proteic peat1 gene of embodiment 6 coding PEAt1 escherichia expression system
The design primer comprises entire reading frame and introduces restriction enzyme site and enzyme is cut the protection base, forward primer 5 '-
CCGGAATTCATGGCCAACCCCCGCATTGAA-3 ' (underscore is the restriction enzyme site of EcoR I) reverse primer 5 '-
CCGCTCGAGCTATATGCTCAGCGCCATGAT-3 ' (underscore is the restriction enzyme site of Xho I), with alternaric bacteria cDNA is template, carry out the RT-PCR amplification with above-mentioned primer, be connected on the PMD18-T cloning vector after the fragment glue that amplification obtains reclaims and check order, whether sequence verification obtains complete gene order and correctly introduces restriction enzyme site, with this plasmid called after PMD18-T-peat1.
PMD18-T-peat1 plasmid and expression vector PET-28-a are carried out the double digestion of EcoR I and Xho I respectively, fragment after enzyme is cut is carried out the glue recovery and is spent the night 16 ℃ of connections, connecting product transforms in BL21 (DE3) expression strain, containing on the solid medium of Kana screening positive clone and extracting plasmid and carry out enzyme and cut evaluation, check order simultaneously and determine that reading frame is correct, the positive colony that is connected with correct peatl gene that obtains is cultivated in containing the liquid nutrient medium of Kana; Treat to induce with chemical inducer IPTG after expression strain grows into logarithmic phase, induced concentration is 1mmol/ml, abduction delivering is centrifugal collection thalline after 12 hours, adopt the method for ultrasonication to extract total protein with purifying buffer B inding Buffer suspension back thalline, carry out SDS-PAGE electrophoresis detection expressing protein.Utilize the Chealting Column post of GE company on AKTA Explorer protein purification instrument, to carry out the purification of samples that purifying obtains purification of recombinant proteins.The purification process of recombinant expression protein is operated according to the ChealtingColumn specification sheets.
The structure and the protein expression of the proteic peat1 gene of embodiment 7 coding PEAt1 pichia yeast expression system
With EcoR I and Xho I difference double digestion PMD18-T-peat1 and pPIC-9K carrier, with the T4 ligase enzyme synthetic gene reorganization is connected pPIC-9K and makes up recombinant vectors pPIC-9K-peat1, heat shock transformed into escherichia coli DH5 α cell, screening positive clone, the PCR method is identified positive colony, to contain the segmental recombinant vectors pPIC-9K-peat1 of correct insertion linearizing through evaluation, electric shocking method transforms pichia spp GS115 cell, coat histidine defect substratum (RDB), the picking transformant is put respectively and is connect MM and MD substratum, screening methyl alcohol responsive type recombinant conversion is inoculated in positive transformant in the 5ml BMG substratum 30 ℃ of 220rmin
-1Cultivate 48h, centrifugal collection thalline adds the resuspended thalline of 10ml BMM substratum, 30 ℃ of 220rmin
-1Continue to cultivate, every 24h adds methyl alcohol 1%, abduction delivering 3-4d, and centrifugal collection supernatant is identified expressing protein with the ELISA method, and makes SDS-PAGE and detect.
Embodiment 8 recombination activation albumen are to the promoter action of wheat seed young root growth
Wheat seed is soaked seed at purification of Recombinant expressing protein 2 μ g.ml
-1The aqueous solution in, with distilled water seed soaking as negative control.Soaking seed after 8 hours is tiled in seed in the culture dish in 28 ℃ of illumination box illumination cultivation, and adds an amount of distilled water to keep humidity.Cultivate random measurement after 7 days and respectively handle the main root length of wheat, each handle measure 30 parallel and average.
The result shows that the purification of Recombinant expressing protein has wheat root growth promoter action is arranged.Root length is being 2 μ g.ml in activator concentration
-1The time, wheat root length has improved 10～15% than control group.
Embodiment 9 recombination activation albumen are to the control action kou of graw mold of tomato
Botrytis cinerea (Botrytis cinerea) is cultivated 7～9d on the PDA substratum, collect spore and make spore suspension.With 2 μ gml
-1Tomato seedling is handled in the spraying of recombinant expression protein extracting solution, compares every processing 30 young plants with clear water.After handling 5d, with botrytis cinerea spore suspension (105ml
-1) inoculate with the spray method challenge, 48h preserves moisture, and carries out the investigation of disease level after the week, calculates prevention effect.The result shows that PEAt1 albumen is handled tomato can strengthen its resistance to gray mold, and prevention effect reaches 65%-70%.
Sequence table
＜110〉Plant Protection institute, Chinese Academy of Agricultral Sciences
＜120〉a kind of protein and encoding gene thereof that improves plants fastness and accelerating vegetation
<130>05-01
<160>1
<170>PatentIn version 3.1
<210>1
<211>624
<212>DNA
＜213〉Alternaria tenuissima bacterium (Alternaria tenuissima)
<400>1
ATGGCCAACC CCCGCATTGA AGAGCTCCCC GACGAGCCCG AGAAGAAGAA CGTCCAGATC 60
GAGGAGGATG AGTCCAGCGA CGAGTCTGAG GGCGAGGAGG GCGAGGTCAG CGTACCCGCG 120
GGCTCCTCCG TCGCTGTCCA CTCCCGCAAC GAGAAGAAGG CTCGCAAGGC CATCGCCAAG 180
CTCGGCCTGA AGCACATCGA CGGCATCACA CGCGTCACCC TCCGCCGACC CAAGAACATC 240
CTCTTTGTCA TCAACCAGCC CGACGTCTAC AAGTCCCCTT CAAGCAACAC CTGGATCATC 300
TTCGGTGAGG CCAAGATCGA GGACCTCAAC TCCCAGGCTC AGGCTTCCGC CGCCCAGCAG 360
CTTGCTCAGG CCGAGGCCGC ATCCCACGAC CACGCCGGCC ACGACCACGG CGACGAGGCC 420
AGCAAGGGCA AGGGCAAGGC TGTCGAGGAC AAGAAGGACG AGGAGGAGGA GGATGACGAT 480
GAGGAGATTG ACGACTCTGG CCTTGAGGCC AAGGACATCG AGCTCGTCAT GCAGCAGGCC 540
AGCGTTTCGC GGAAGAAGGC CGTCAAGGCC CTCAAGGAGA ACGATAACGA TATAGTCAAC 600
TCCATCATGG CGCTGAGCAT ATAG 624
Met Ala Asn Pro Arg Ile Glu Glu Leu Pro Asp Glu Pro Glu Lys Lys
1 5 10 15
Asn Val Gln Ile Glu Glu Asp Glu Ser Ser Asp Glu Ser Glu Gly Glu
20 25 30
Glu Gly Glu Val Ser Val Pro Ala Gly Ser Ser Val Ala Val His Ser
35 40 45
Arg Asn Glu Lys Lys Ala Arg Lys Ala Ile Ala Lys Leu Gly Leu Lys
50 55 60
His Ile Asp Gly Ile Thr Arg Val Thr Leu Arg Arg Pro Lys Asn Ile
65 70 75 80
Leu Phe Val Ile Asn Gln Pro Asp Val Tyr Lys Ser Pro Ser Ser Asn
85 90 95
Thr Trp Ile Ile Phe Gly Glu Ala Lys Ile Glu Asp Leu Asn Ser Gln
100 105 110
Ala Gln Ala Ser Ala Ala Gln Gln Leu Ala Gln Ala Glu Ala Ala Ser
115 120 125
His Asp His Ala Gly His Asp His Gly Asp Glu Ala Ser Lys Gly Lys
130 135 140
Gly Lys Ala Val Glu Asp Lys Lys Asp Glu Glu Glu Glu Asp Asp Asp
145 150 155 160
Glu Glu Ile Asp Asp Ser Gly Leu Glu Ala Lys Asp Ile Glu Leu Val
165 170 175
Met Gln Gln Ala Ser Val Ser Arg Lys Lys Ala Val Lys Ala Leu Lys
180 185 190
Glu Asn Asp Asn Asp Ile Val Asn Ser Ile Met Ala Leu Ser Ile
195 200 205
Claims (9)
1. one kind is improved disease resistance of plant and the protein that promotes plant-growth, and its sequence is shown in SEQ ID NO1, and this protein source is in alternaric bacteria (Alternaria).
2. the protein of raising disease resistance of plant according to claim 1 and promotion plant-growth is used to prepare raw product or the highly finished product that promote plant-growth and improve plant resistance to environment stress.
3. the purposes that the protein of raising disease resistance of plant according to claim 2 and promotion plant-growth, described goods are used to improve plant resistance to environment stress and promote plant-growth.
4. according to the proteinic aminoacid sequence in the claim 1, obtain this proteinic nucleotide sequence of coding from alternaric bacteria, its sequence is shown in SEQ ID NO1.
5. a recombinant plasmid contains the described nucleotide sequence of claim 4.
6. a transformant contains the host microorganism cell that imports the described recombinant plasmid of claim 5.
7. the transformant in the claim 6, the host microorganism cell is optional from intestinal bacteria and Pichia yeast.
8. with the recombinant protein goods of the described transformant preparation of claim 6, comprise raw product or highly finished product.
9. the purposes that is used to improve disease resistance of plant and promotes plant-growth of the recombinant protein goods in according to Claim 8.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102204571A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of Alternaria alternata metabolic product in controlling watermelon fusarium oxysporum |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN108522508A (en) * | 2018-04-18 | 2018-09-14 | 中国农业科学院植物保护研究所 | Applications of the protein elicitor PeaT1 in inducing the anti-aphid of crops |
| CN110205248A (en) * | 2019-06-12 | 2019-09-06 | 中国矿业大学(北京) | A kind of method of simultaneous inoculation AM and the promotion plant growth of DSE fungi and its microbial bacterial agent used |
| CN113913441A (en) * | 2021-12-07 | 2022-01-11 | 湖南科技学院 | Application of rice nascent polypeptide binding complex alpha subunit NACA gene in plant osmotic stress resistance |
| CN114437231A (en) * | 2020-11-02 | 2022-05-06 | 苏州乙水茉生物科技有限公司 | Bivalent plant immune protein AB-NAC and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1142179C (en) * | 2001-10-09 | 2004-03-17 | 邱德文 | Multifunctional mycoprotein product for plant and its prepn |
-
2006
- 2006-09-27 CN CN200610152700A patent/CN101153057B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102204571A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of Alternaria alternata metabolic product in controlling watermelon fusarium oxysporum |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN108118004B (en) * | 2017-12-15 | 2021-06-25 | 北京工商大学 | Application of pichia stipitis in preventing and treating postharvest diseases of fruits |
| CN108522508A (en) * | 2018-04-18 | 2018-09-14 | 中国农业科学院植物保护研究所 | Applications of the protein elicitor PeaT1 in inducing the anti-aphid of crops |
| CN110205248A (en) * | 2019-06-12 | 2019-09-06 | 中国矿业大学(北京) | A kind of method of simultaneous inoculation AM and the promotion plant growth of DSE fungi and its microbial bacterial agent used |
| CN114437231A (en) * | 2020-11-02 | 2022-05-06 | 苏州乙水茉生物科技有限公司 | Bivalent plant immune protein AB-NAC and application thereof |
| CN113913441A (en) * | 2021-12-07 | 2022-01-11 | 湖南科技学院 | Application of rice nascent polypeptide binding complex alpha subunit NACA gene in plant osmotic stress resistance |
| CN113913441B (en) * | 2021-12-07 | 2023-08-04 | 湖南科技学院 | Application of rice nascent polypeptide binding complex alpha subunit NACA gene in osmotic stress resistance of plants |
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